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Title Regulation of myo-inositol biosynthesis by p53-ISYNA1 pathway( 本文 )
Author(s) 胡口, 智之
Citation
Issue Date 2017-03-24
URL http://ir.fmu.ac.jp/dspace/handle/123456789/964
Rights Fulltext: Published version is "Int J Oncol. 2016
Jun;48(6):2415-24. doi: 10.3892/ijo.2016.3456. Published by Spandidos Publications".
DOI
Text Version ETD
1
Regulation of myo-inositol biosynthesis by p53-ISYNA1 pathway.
（ 新規
p53
下流遺伝子ISYNA1
によるミオイノシトール合成制御 ）Tomoyuki Koguchi
胡 口 智 之Department of Urology, The Graduate School of Medicine, Fukushima Medical University
福 島 県 立 医 科 大 学 大 学 院 医 学 研 究 科 泌 尿 器 外 科 学 分 野
March, 2017
2 Abstract
In response to various cellular stresses, p53 exerts its tumor suppressive effects such as
apoptosis, cell cycle arrest, and senescence through the induction of its target genes.
Recently, p53 was shown to control cellular homeostasis by regulating energy metabolism,
glycolysis, antioxidant effect, and autophagy. However, its function in inositol synthesis was
not reported so far. Through a microarray screening, I found that five genes related with
myo-inositol metabolism were induced by p53. DNA damage enhanced intracellular myo-
inositol content in HCT116 p53+/+ cells, but not in HCT116 p53-/- cells. I also indicated that
inositol 3-phosphate synthase 1 (ISYNA1) which encodes an enzyme essential for myo-
inositol biosynthesis as a direct target of p53. Activated p53 regulated ISYNA1 expression
through p53 response element in the seventh exon. Ectopic ISYNA1 expression increased
myo-inositol levels in the cells and suppressed tumor cell growth. Knockdown of ISYNA1
caused resistance to adriamycin treatment, demonstrating the role of ISYNA1 in p53-
mediated growth suppression. Furthermore, ISYNA1 expression was significantly
associated with p53 mutation in bladder, breast cancer, head and neck squamous cell
carcinoma, lung squamous cell carcinoma, and pancreatic adenocarcinoma. Our findings
revealed a novel role of p53 in myo-inositol biosynthesis which could be a potential
therapeutic target.
3 Introduction
p53 is one of the most frequently mutated tumor suppresser genes (1, 2). In response to
various cellular stresses, ATM-Chk2 cascade stabilizes p53 protein through the
phosphorylation of its N-terminal domain (3). Activated p53 functions as a transcription
factor and exerts its tumor suppressive effects such as apoptosis, cell cycle arrest, and
senescence through the induction of its target genes (1, 2). In addition to genes related with
cell proliferation, regulation of glycolysis (4), energy metabolism, antioxidant effect (5),
autophagy (6), and respiration with mitochondria are reported as novel functions of p53.
Thus, p53 regulates not only tumor cell growth but also pathways related with cellular
homeostasis. Since inactivation of p53 is the most common feature of cancer cells, the
elucidation of p53 signaling pathways would contribute to the understanding of tumor cells
as well as for drug development.
Myo-inositol is water-soluble vitamin found in a variety of food products, and are also
synthesized in cells (7). Previous studies indicated that myo-inositol has various functions
including glucose and lipid metabolism (8, 9), neurotropic effect (10), and tumor
suppression (11-13). However, the regulation of myo-inositol biosynthesis in cancer tissues
has not been disclosed yet. Through a cDNA microarray screening using mRNAs isolated
from HCT116 p53+/+ and HCT116 p53-/- cells, here I identified ISYNA1 which encodes an
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enzyme essential for myo-inositol biosynthesis as a novel p53 target.
5 Materials and Methods
cDNA microarray
Gene expression analysis was performed using SurePrint G3 Human GE 8x60K microarray
(Agilent, Santa Clara, CA, USA) according to the manufacturer's protocol. Briefly, HCT116
p53+/+ or HCT116 p53-/- cells were treated with 2 µg/ml of adriamycin (ADR) for 2 h and
incubated at 37°C until harvest. At 12 h, 24 h and 48 h after treatment, total RNA was
isolated from the cells using standard protocols. Each RNA sample was labeled and
hybridized to array slides.
Cell culture and treatment.
Human embryonic kidney cells HEK293T were obtained from Riken Cell Bank (Riken Cell
Bank, Ibaraki, Japan). Human cancer cell lines U373MG (astrocytoma), HepG2
(hepatocellular carcinoma), and HCT116 (colorectal adenocarcinoma) were purchased
from American Type Culture Collection (ATCC, Manassas, VA, USA). HCT116 p53+/+ and
HCT116 p53-/- cells lines were gifts from B. Vogelstein (Johns Hopkins University,
Baltimore, MD, USA). HEK293T, HCT116, and HepG2 cells were transfected with plasmids
using Fugene6 (Promega, Madison, WI, USA). U373 MG cells were transfected with
plasmids using Fugene6 or Lipofectamin LTX (Invitrogen, Carlsbad, CA, USA). Small
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interfering RNA (siRNA) oligonucleotides, commercially synthesized by Sigma Genosys,
were transfected with Lipofectamine RNAiMAX reagent (Invitrogen). Sequences of siRNA
oligonucleotides are shown in Table 1. I generated and purified replication-deficient
recombinant viruses expressing p53 (Ad-p53) or LacZ (Ad-LacZ) as described previously
(14). U373MG (p53-mutant) cells were infected with viral solutions at various amounts of
multiplicity of infection (MOI) and incubated at 37°C until the time of harvest. For treatment
with genotoxic stress, cells were incubated with 2 g/ml of ADR for 2 h.
Plasmid construction.
The entire coding sequence of ISYNA1 isoform1 and isoform4 were amplified by PCR
using KOD-Plus DNA polymerase (Toyobo, Osaka, Japan), and inserted into the EcoRV
and XhoI sites of pCAGGS vector. ISYNA1 isoform2 expression vector was constructed by
site-directed mutagenesis using ISYNA1 isoform 1 as a template. The construct was
confirmed by DNA sequence analysis. Primers are shown in Table 1.
Quantitative real-time PCR.
Total RNA was isolated from human cells and mouse tissues using RNeasy Plus Mini Kits
(Qiagen, Valencia, CA, USA) and RNeasy Plus Universal Mini Kits (Qiagen, Valencia, CA,
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USA) according to the manufacturer’s instructions. Complementary DNAs were
synthesized using Super Script III reverse transcriptase (Invitrogen). Quantitative real-
timePCR (qPCR) was conducted using SYBR Green Master Mix on a Light Cycler 480
(Roche, Basel, Switzerland). Primer sequences are shown in Table 1.
Western blot analysis
To prepare whole cell extracts, cells were collected and lysed in chilled RIPA buffer (50
mmol/L Tris-HCl at pH 8.0, 150 mmol/L NaCl, 0.1% SDS, 0.5% sodium deoxycholate, and
1% NP40) containing 1 mM phenyl methylsulphonyl fluoride (PMSF), 0.1 mM
dithiothreitol
(DTT) and 0.1% Calbiochem Protease Inhibitor Cocktail Set III, EDTA-Free (EMD
Chemicals Inc., Merck KGaA, Darmstadt, Germany). Samples were sonicated for 15 min
with a 30-sec on/30-sec off cycle using Bioruptor UCD-200 (Cosmobio, Tokyo, Japan). After
centrifugation at 16,000 × g for 15 min, supernatants were collected and boiled in SDS
sample buffer (Biorad, Hercules, CA, USA). SDS-polyacrylamide gel electrophoresis (SDS-
PAGE) was performed using with 12% acrylamide separating gel for each sample, and the
proteins were then transferred to a nitrocellulose membrane (Hybond™ ECL™, Amersham,
Piscataway, NJ, USA). Protein bands on western blots were visualized by
chemiluminescent detection (ECL, Amersham and Immobilon, Millipore). Anti-β-actin
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monoclonal antibody (AC-15) was purchased from Abcam (Cambridge, UK). Anti- ISYNA1 monoclonal antibody (sc-271830) and anti-p53 monoclonal antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-p21WAF1 monoclonal antibody (OP64) was purchased from Merck Millipore (Darmstadt, Germany).
Immunofluorescence microscopy
Cells were seeded on coverslips in 24-well plates. After each treatment indicated in the
text, cells were washed in phosphate-buffered saline (PBS) before fixation in 4%
paraformaldehyde. Cells were immunostained overnight with primary antibodies followed
by incubation with Alexa Fluor 488-conjugated secondary IgG (Molecular Probes) for 1 h.
Cells were subject to 4'-6-Diamidino-2-phenylindole (DAPI) staining to visualize cell nuclei.
Immunofluorescence was visualized and recorded on an Olympus FV1000D laser confocal
microscope. Images were processed using Olympus FV10-ASW software and Adobe
Photoshop CS3.
Gene reporter assay
DNA fragments, including the potential p53-response elements (REs), were amplified and
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subcloned into the pGL4.24 vector (Promega). Point mutations “T” were inserted at the 4th
and the 14th nucleotide “C” and the 7th and the 17th nucleotide “G” of each RE by site-
directed mutagenesis. Reporter assays were performed using the Dual Luciferase assay
system (Promega) as described previously (15). Primers for amplification and mutagenesis
are shown in Table 1.
Chromatin immunoprecipitation (ChIP) assay.
ChIP assay was performed using EZ-Magna ChIP G Chromatin Immunoprecipitation Kit
(Merck Millipore, Darmstadt, Germany) following the manufacturer’s protocol. In brief,
U373MG cells infected with Ad-p53- or Ad-LacZ at an MOI of 10 were cross-linked with 1%
formaldehyde for 10 min, washed with PBS, and lysed in nuclear lysis buffer. The lysate
was then sonicated using Bioruptor UCD-200 (CosmoBio) to shear DNA to approximately
200-1000 bp. Supernatant from 1 × 106 cells was used for each immunoprecipitation with
anti-p53 antibody (OP140, Merck Millipore) or normal mouse IgG (sc-2025, Santa Cruz,
Santa Cruz, CA, USA). Column-purified DNA was quantified by qPCR. Primer sequences
are shown in Table 1.
10 Myo-inositol (MI) assay
To prepare cell homogenate, cells were collected and suspended in PBS. Samples were
sonicated for 15 min with a 30-sec on/30-sec off cycle using Bioruptor UCD-200
(Cosmobio, Tokyo, Japan). After centrifugation at 16,000 × g for 5 min, myo-inositol content
in supernatants was measured using myo-Inositol assay kit (K-INOSL, Megazyme
International Ireland, Bray, Wicklow, Ireland) according to the manufacturer’s instruction.
Colony formation assay
HCT116 cells and HepG2 cells were seeded on 6-well flat bottomed microplates. At 24 h
after seeding, cells were transfected with pCAGGS (Mock) vector or pCAGGS/ISYNA1
isoform1. HCT116 and HepG2 cells were cultured with 0.5 mg/ml or 1.2 mg/ml of G418,
respectively. After 2 or 3 weeks of drug selection, colonies were washed in phosphate
buffered saline and stained with 0.1% crystal violet for 1 day.
ATP assay
HCT116 p53+/+ cells were transfected with siRNAs and seeded on 24 well plates. At 24 h
after transfection, cells were treated with 2 µg/ml of ADR for 2 h. At 48 h after ADR
treatment, cell viability was evaluated by Cell Titer-Glo Luminescent Cell Viability Assay
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(Promega). After removal of culture medium, cells were incubated with 100µl of Cell Titer-
Glo Reagent and 100µl of culture medium for 10 minutes and lysed. The luminescence of
cell lysate was measured by ARVO X3 plate reader (Perkin Elmer, Waltham, MA, USA)
according to the manufacturer's protocol.
Animal models
p53-/- mice were provided by RIKEN BioResource Center (Ibaraki, Japan) (16). All mice were maintained under specific pathogen-free conditions and were handled in
accordance with the Guidelines for Animal Experiments of the University of Tokyo.
p53+/+ and p53-/- mice at 6 weeks of age were irradiated with 10Gy of X-ray. At 24 h after irradiation, mice were sacrificed for liver extraction. The experiment was
conducted after the approval of the Animal Experiment Committee of Institute of Medical
Science, The University of Tokyo, Tokyo, Japan.
Database analysis
ISYNA1 expression and p53 mutation status in clinical samples were obtained from the TCGA project via data portal on 15 May 2015 (17). The association between ISYNA1
expression and the presence of the p53 gene mutation was determined by using the
12 Student's t-test.
13 Result
p53 regulates genes related with myo-inositol metabolism
To screen novel p53 target genes, I conducted cDNA microarray analysis using mRNAs
isolated from HCT116 p53+/+ and HCT116 p53-/- cells that were treated with 2 g/ml of
adriamycin (ADR). Figure 1A shows a schematic representation of inositol phosphate
metabolism pathway. The result of cDNA microarray analysis indicated that five genes
related with myo-inositol metabolism were induced by p53 (Fig. 1B). I selected inositol 3-
phosphate synthase (ISYNA1) for further analysis, because ISYNA1 showed the highest
expression among the five genes.
To validate the result of cDNA microarray analysis, I performed quantitative real-time
PCR (qPCR) analysis and western blotting of ISYNA1 using HCT116 p53+/+ and HCT116
p53-/- cells treated with ADR. As a result, I found dose-dependent induction of ISYNA1
mRNA and protein only in HCT116 p53+/+ cells in response to ADR treatment (Fig. 1C). I
also confirmed the induction of ISYNA1 mRNA and protein by ADR treatment in HepG2
(Fig. 1D). Moreover, transfection with siRNA against p53 remarkably inhibited the induction
of ISYNA1 (Fig. 1E). p53-mediated induction of ISYNA1 was also observed in U373MG
glioblastoma cells that were infected with adenovirus designed to express wild-type p53
(Ad-p53) (Fig. 1F). These results clearly indicated that ISYNA1 was regulated by p53.
14 Expression and subcellular localization of ISYNA1
There are three major variants of human ISYNA1, namely isoform 1, 2, and 4. All isoforms
are similar in domain structure as shown in Figure 2A. I constructed plasmids expressing
each isoform. Result of western blotting indicated that isoform 1 is the major ISYNA1
isoform that was expressed in HCT116 and HepG2 cells treated with ADR (Fig. 2B,C).
Then I performed immunocytochemical analysis using HCT116 p53+/+, HCT116 p53-/-
cells, or HepG2 cells (Fig. 2D,E). ADR treatment increased ISYNA1 protein in the
cytoplasm and the nucleus of HCT116 p53+/+ and HepG2 cells, but ISYNA1 expression was
very low in HCT116 p53-/- cells or HepG2 cells treated with sip53.
Identification of ISYNA1 as a novel p53 target.
To investigate whether ISYNA1 is a direct target of p53, I searched for p53 response
element (RE) (18) within the ISYNA1 genomic region which is located on Chromosome
19p13. I found putative p53 RE in the promoter region (RE1) and the seventh exon (RE2)
(Fig. 3A). I subcloned DNA fragments including the RE1 or RE2 into pGL4.24 vector
(pGL4.24/RE1 and pGL4.24/RE2) and performed gene reporter assay using U373MG cells.
As a result, U373MG cells transfected with pGL4.24/RE1 or pGL4.24/RE2 showed
enhanced luciferase activity only in the presence of plasmid expressing wild-type p53 (Fig.
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3B). In addition, base substitutions within the RE1 and RE2 (pGL4.24/RE1mt and
pGL4.24/RE2mt) completely abolished the enhancement of luciferase activity (Fig. 3B). To
investigate whether p53 could directly bind to RE2 which showed higher transcriptional
activatity, I performed chromatin immunoprecipitation (ChIP) assay using U373MG cells
that were infected with Ad-p53 or Ad-LacZ. qPCR analysis of the immunoprecipitated DNA
indicated that the p53 protein bound to the genomic fragment that included the RE2 (Fig.
3C). Taken together, p53 directly regulated ISYNA1 expression through binding to the RE2
in the seventh exon.
Growth suppressive effect of ISYNA1
ISYNA1 is the rate-limiting enzyme of myo-inositol de novo synthesis (7) which is
conserved among eukaryotes (19-25). To evaluate the biosynthesis of myo-inositol by
ISYNA1, I performed myo-inositol (MI) assay using 293T cells that were transfected with
mock or plasmid expressing mock or ISYNA1 isoform 1 (Fig. 4A). The results showed that
intracellular myo-inositol content in cells expressing ISYNA1 isoform 1 was significantly
higher than those in control cells. In addition, DNA damage significantly increased
intracellular myo-inositol content in HCT116 p53+/+ cells, but did not affect the myo-inositol
content in HCT116 p53-/- cells (Fig. 4B). Thus, our results indicated that p53 could regulate
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intracellular myo-inositol levels in response to DNA damage.
I also evaluated the effect of p53-ISYNA1 pathway on cancer cell growth. The result of
colony formation assay using HCT116 and HepG2 cells indicated that ISYNA1
overexpression suppressed cell proliferation (Fig. 4C). I then designed three siRNAs (siA,
siB and siC) and found that siRNAs effectively suppressed ISYNA1 mRNA and protein (Fig.
4D). I performed ATP assay using HCT116 p53+/+ cells and found that ISYNA1-silencing
caused resistance to ADR treatment (Fig. 4E). These results indicated ISYNA1 is likely to
be one of the key mediators of p53 induced growth suppression.
Regulation of ISYNA1 by p53 in vivo.
Since ISYNA1 is conserved among eukaryotes, I investigated whether mouse Isyna1 is
also regulated by p53. p53 wild-type or p53 knockout mice at 6 weeks of age were
irradiated with 10 Gy of X-ray. At 24 h after irradiation, I isolated total RNA from liver
tissues. qPCR analysis revealed that mouse Isyna1 mRNA was induced by DNA damage
only in p53 wild type mice (Fig. 5A). Screening of p53 RE within Isyna1 genomic region
identified a putative RE (mRE) at about 10 kb upstream of the Isyna1 gene (Fig. 5B). I
subcloned a DNA fragment including mRE into the pGL4.24 vector (pGL4.24/mRE) and
performed gene reporter assay using U373MG cells (Fig. 5C). Luciferase activity was
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strongly enhanced by co-transfection with wild-type p53 but not by that with mutant p53. In
addition, base substitutions within mRE diminished the enhancement of luciferase activity,
demonstrating regulation of Isyna1 by p53 through mRE.
I also analyzed whether p53 regulates ISYNA1 in human cancer tissues. Correlation
between p53 mutation and ISYNA1 expression was analyzed by using omics data of
various tumor tissues released from the TCGA database (17). Interestingly, ISYNA1 mRNA
expression in bladder cancer, breast cancer, head and neck squamous cell carcinoma, lung
squamous cell carcinoma, and pancreatic adenocarcinoma was significantly decreased in
tumor tissues with p53 mutation compared with those without p53 mutation (Fig. 5D).
These findings indicate that p53 regulates ISYNA1 expression in vivo.
18 Discussion
ISYNA1 is a conserved gene among eukaryotes; fungus, plants, insects and vertebrates.
Previous reports showed that ISYNA1 was regulated by E2F1 (26), myo-inositol phosphate
synthase ( MIPS, as the homolog of ISYNA1 in plants) (27). One study reported that MIPS
probably can control chromatin remodeler (ATRX) activity to stop the spreading of histone
methylation (27). But, very little is known about the regulation of ISYNA1. Here I identified
ISYNA1 as a novel p53 target. ISYNA1 is a key enzyme which affects myo-inositol de novo
synthesis (7, 28, 29). In addition, p53 induced INPP1 and INPP5 (30) that are involved in
myo-inositol salvage pathway.
Myo-inositol is one of the chemical compounds which is essential for living organisms (31),
and myo-inositol depletion affects cell survival and growth (32). Myo-inositol was also
reported to suppress tumor growth in vitro and in vivo (33-40). Previous studies indicated
that myo-inositol suppresses phosphorylation of Akt and Erk by inhibiting PI3K activity (12,
13). p53 was also shown to suppress PI3K-Akt pathway by inducing PTEN (41) and Phlda3
(42). Our results suggested a novel mechanism whereby p53 negatively regulates PI3K-Akt
pathway by inducing ISYNA1.
Epidemiological studies indicate that myo-inositol prevents progression of dysplasia in
smokers (11-13), and decreases tumorigenesis in chronic hepatitis patients (35). These
19
findings suggested that p53 would suppress tumorigenesis by inducing biosynthesis of
myo-inositol. I also found that ISYNA1 was induced in mice liver tissue by DNA damage. To
evaluate the chemopreventive effect of myo-inositol, I fed p53 knockout mice with myo-
inositol in drinking water. However, oral myo-inositol did not suppress tumor development
(Supplement 1). Although, myo-inositol was shown to suppress liver cancer (34, 35), liver
cancer is relatively rare for p53 knockout mice compared with lymphoma of thymus or
spleen (43). In addition, although induction of Isyna1 was observed in liver tissues, Isyna1
was not induced in thymus and spleen (data not shown). Therefore, to evaluate the
chemopreventive effect of myo-inositol or ISYNA1 in vivo, liver cancer model would be
appropriate. Also, I did not evaluate that myo-inositol circulated in the cell as the part of
phosphatidylinositol system. Consequently, I thought major limitation of this study is the
unidentified correlation between function of myo-inositol and p53 target genes.
Taken together, ISYNA1 was shown to be a mediator of p53 dependent growth
suppression, and ISYNA1 expression was reduced in several types of cancers with p53
mutations. Therefore, myo-inositol could be a potential anti-cancer agent for cancer cells
with p53 mutation. Our findings revealed a novel role of p53 in myo-inositol biosynthesis
which could be a possible therapeutic target.
20 Acknowledgement
I thank Prof. Yoshiyuki Kojima and Dr. Kei Ishibashi from our department for suggestions. I
thank Prof. Koichi Matsuda and Dr. Chizu Tanikawa from Laboratory of Clinical Sequence,
Department of Computational biology and medical Sciences, Graduate school of Frontier
Sciences, The University of Tokyo for suggestions and encouragement. Dr. Jinichi Mori for
discussion. Satomi Takahashi and Misato Oshima for technical assistance. I also thank The
Cancer Genome Atlas (TCGA) project and members of the Cancer Genomics Hub
(CGHub) for making all TCGA data publicly accessible. This work was supported partially
by grant from Japan Society for the Promotion of Science and Ministry of education,
culture, sports, science and technology of Japan to K.M and C.T., grant from Japan Agency
for medical Research and Development to K.M. and C. T., grant from the Ministry of Health,
Labour and Welfare, Japan to K.M., and grant in-Aid from the Tokyo Biochemical Research
Foundation to K.M.
Disclosure statement
The author declares no conflicts of Interest.
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24 Figures and Tables
Table1: Sequence of primers and oligonucleotides.
25 Figure 1: Regulation of ISYNA1 by p53
26 Figure 1: Regulation of ISYNA1 by p53
(A) Schematic representation of the inositol phosphate metabolism pathway.
PI:phosphatidylinositol, PIP:phosphatidylinositol 4-phosphate , PIP2:phosphatidylinositol 4,5-bisphosphate, Ins(1,4,5)P3:inositol 1,4,5-trisphosphate, Ins(1,4)P2:inositol 1,4- bisphosphate, Ins(4)P:inositol 4-phosphate, Ins(1,3,4,5)P4:inositol 1,3,4,5-
tetrakisphosphate, Ins(1,3,4)P3:inositol 1,3,4-trisphosphate, Ins(3,4)P2:inositol 3,4- phosphtate, Ins(3)P:inositol 3-phosphate, Ins(1,3)P2:inositol 1,3-bisphosphate, Ins(1)P:inositol 1-phosphate, G6P:glucose 6-phosphate, DAG:diacylglycerol,
PA:phosphatidate, CDP-DAG: CDP-diacylglycerol (B) Induction of genes related with myo- inositol biosynthesis by p53. HCT116 p53+/+ and HCT116 p53-/- cells were treated with 2 µg/ml of adriamycin (ADR) for 2 h. mRNAs isolated from these cells were subjected to microarray analysis. Five genes related with inositol phosphate metabolism were shown to be induced by p53. (C) qPCR analysis (upper) and western blotting (lower) of ISYNA1, p53, and WAF1 in HCT116 p53+/+ and HCT116 p53-/- cells at 36 h after treatment with ADR for 2 h. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and -actin were used for the normalization of expression levels. Error bars represent S.D. (n = 3). (D) qPCR analysis (upper) and western blotting (lower) of ISYNA1, p53, and WAF1 in HepG2 cells at 36 h after treatment with ADR for 2 h. GAPDH and β-actin were used for the normalization of expression levels. Error bars represent S.D. (n = 3). (E, F) qPCR analysis of ISYNA1 mRNA in HepG2 (E) or U373MG (F) cells. At 24 h after transfection of each siRNA, HepG2 cells were treated with 2 µg/ml of ADR for 2 h. At 40 h after treatment, cells were harvested for qPCR analysis. U373MG cells were harvested at 36 h after infection with Ad-p53.
siEGFP or Ad-LacZ were used as controls. GAPDH was used for the normalization of expression levels. Error bars represent S.D. (n = 3). The P value was calculated by
27 Student’s t-test.
28 Figure 2: Expression and localization of ISYNA1
29 Figure 2: Expression and localization of ISYNA1
(A) Upper: genomic structure of ISYNA1 variants. Black boxes indicate the locations and relative sizes of exons. Lower: Domain structure of ISYNA1 isoforms. (B) Western blotting of ISYNA1, p53, and WAF1 at 36 h after treatment with 2 µg/ml of adriamycin (ADR) for 2 h in HCT116 p53+/+ and HCT116 p53-/- cells. HEK293T cells transfected with plasmid
designed to express ISYNA1 isoform 1, 2, 4 were used for molecular weight estimation of endogenous ISYNA1 protein. β-actin was used for the normalization of expression levels.
(C, E) At 24 h after transfection of each siRNA, HepG2 cells were treated with 2 µg/ml of ADR for 2 h. At 40 h after treatment, ISYNA1 expression was evaluated by (C) western blotting or (E) immunocytochemistry with an anti-ISYNA1 antibody (Alexa Fluor 488;
green). Expression of p53, WAF1, and β-actin was also shown. DAPI was used to visualize the nuclei (blue). (D) Immunocytochemical analysis of ISYNA1 with an anti-ISYNA1
antibody (Alexa Fluor 488; green) at 36 h after treatment with 2 µg/ml of ADR for 2 h in HCT116 p53+/+ and HCT116 p53-/- cells. DAPI was used to visualize the nuclei (blue).
30
Figure 3: Identification of ISYNA1 as a novel p53 target.
31
Figure 3: Identification of ISYNA1 as a novel p53 target.
(A) Upper: genomic structure of human ISYNA1. Black boxes indicate the locations and relative sizes of exons. White boxes indicate the locations of p53 response elements (RE).
Lower: Comparison of two REs to the consensus p53 RE. R, purine; W, A or T; Y
pyrimidine. Identical nucleotides to the consensus sequence are written in capital letters.
The underlined cytosine and guanine were substituted for thymine to examine the
specificity of the p53-binding site. (B) Luciferase assay of REs with or without mutations in the RE by using U373MG cells. Luciferase activity is indicated relative to the activity of the mock vectors. The plasmid expressing p53 carrying a missense mutation (R175H) served as a negative control. Error bars represent S.D. (n = 3). (C) ChIP assay was performed using U373MG cells that were infected with Ad-p53 (lane 2-4) or Ad-LacZ (lane 1) at an MOI of 10. DNA-protein complexes were immunoprecipitated with an anti-p53 antibody (lanes 1 and 4) followed by qPCR analysis. Immunoprecipitates with a normal IgG (lane 3) or in the absence of an antibody (lane 2) were used as negative controls. Error bars, S.D.
(n = 3).
32
Figure 4: Regulation of myo-inositol synthesis and cell growth by p53-ISYNA1 pathway
33
Figure 4: Regulation of myo-inositol synthesis and cell growth by p53-ISYNA1
pathway
(A) At 36 h after transfection with mock vector or plasmid expressing ISYNA1 isoform1, the amounts of myo-inositol were evaluated. Total protein content was used for normalization.
Error bars, S.D. (n = 3). (B) Upper: myo-inositol assay at 36 h after treatment with 2 µg/ml of ADR in HCT116 p53+/+ and HCT116 p53-/- cells. Total protein content was used for normalization. Error bars, S.D. (n = 3). Lower: Expression of ISYNA1 and p53 protein. (C) HCT116 and HepG2 cells were transfected with mock or plasmid expressing ISYNA1 isoform 1. The number of colonies was quantified by Image J software. Error bar, S.D. (n = 3). (D) At 24 h after transfection of each siRNA, HCT116 p53+/+ cells were treated with 2 µg/ml of ADR for 2 h. At 48 h after treatment, qPCR (upper) and western blot (lower) analyses were performed to evaluate the expression of ISYNA1 and p53. siEGFP was used as a control. GAPDH and β-actin were used for the normalization of expression levels. Error bars represent S.D. (n = 3). (E) At 24 h after transfection of each siRNA, HCT116 p53+/+ cells were treated with 2 µg/ml of ADR for 2 h. At 48 h after treatment, ATP assay was performed. Relative cell viability was calculated by dividing the luminescence of ADR-treated cells by that of untreated cells. Error bars represent S.D. (n = 3).
34 Figure 5: Regulation of ISYNA1 by p53 in vivo.
35 Figure 5: Regulation of ISYNA1 by p53 in vivo.
(A) qPCR analysis of Isyna1 in mouse livers. Mice were divided into four groups; p53 wild type mice without irradiation (W), p53 wild type mice with irradiation (WX), p53 knockout mice without irradiation (K), p53 knockout mice with irradiation (KX) (n = 6 per group).
Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) was used for the normalization of
expression level. Top bar represents maximum observation, lower bar represents minimum observation, the top side of the box represents the third quartile, and the bottom side, the first quartile. The middle bar represents the median value. The P value was calculated by Student’s t-test. (B) Upper: genomic structure of mouse Isyna1. Black boxes indicate the locations and relative sizes of exons. The white box indicates the location of the p53 response element (mRE). Lower: Comparison of mRE to the consensus p53RE. R, purine;
W, A or T; Y pyrimidine. (C) Luciferase assay of mRE in U373MG with or without mutation of the RE. Luciferase activity is indicated relative to the activity of the mock vectors. The plasmid expressing mouse p53 carrying a missense mutation (R172H) served as a negative control. Error bars represent the S.D. (n = 3). (D) Box plot of ISYNA1 expression in bladder cancer, breast cancer, head and neck squamous cell carcinoma, lung squamous cell carcinoma, and pancreatic adenocarcinoma tissues from the TCGA database. The vertical axis indicates the normalized expression level of ISYNA1, top bar represents maximum observation, lower bar represents minimum observation, the top side of the box represents the third quartile, and the bottom side, the first quartile. The middle bar
represents the median value. The P value was calculated by Student’s t-test.
